We propose studies on three related bacterial exotoxins -diphtheria toxin, exotoxin A from Pseudomonas aeruginosa (Pa toxin), and choleragen - all of which catalyze NAD ion-dependent ADP-ribosylation reactions. With diphtheria toxin we propose: to characterize differences between NAD ion-binding and non-binding forms; to collaborate in X-ray crystallographic studies of Fragment A; to use affinity methods to isolate active-site peptides; and to use crosslinked conjugates containing Fragment A as an approach to the mode of entry to toxins into mammalian cells. With Pa toxin we will concentrate on elucidating structure-activity relationships, including the relationship of a 26,000 dalton, enzymically active peptide to the whole toxin, and will study possible approaches to studying cell surface attachment. With choleragen we will employ our newly developed mutant screening procedure (ganglioside filter assay) to isolate mutants of V. cholerae altered in structural and control genes for choleragen production and excretion. The mutants will be analyzed biochemically, genetically, and for possible use in developing a live oral vaccine against cholera. Photoactivatable, heterobifunctional reagents will be used to probe associations of choleragen A an B chains after cell surface attachment.